BMP6 stimulation increases internalization of VE-cadherin. (A) BMP6 increases the internalization rate of cell-surface-labeled VE-cadherin. HUVECs were incubated with the VE-cadherin extracellular domain-targeting antibody BV6 at 4°C. VE-cadherin internalization was monitored by uptake of BV6 antibody upon growth factor treatment for 60 min at 37°C. Remaining cell-surface antibodies, visible in ‘no acid wash’ conditions, were washed away with a mild acid solution and internalized VE-cadherin antibodies were visualized in fixed cells by addition of a fluorophore-coupled secondary antibody (green). Blue, DAPI. Scale bars: 20 µm. (B) Quantitation of VE-cadherin vesicles visible in A, normalized to untreated control cells. Mean±s.d. from three independent experiments. (C) BMP6 triggers internalization of cell-surface biotinylated VE-cadherin. HUVEC surface proteins were biotinylated at 4°C and growth factor-induced endocytosis was allowed to occur for 60 min at 37°C. Subsequently, remaining cell-surface biotin, which is visible in 4°C versus 4°C stripped samples, was stripped at 4°C and cells were rinsed and solubilized. Internalized biotinylated proteins were subjected to immunoprecipitation and samples were blotted as indicated. Total cell lysates (TCLs) represent samples before precipitation. Dotted line indicates merge of the same, but differentially exposed blot (left, 10 s; right, 60 s). (D) Quantitation of VE-cadherin signal intensities depicted in C and normalized to TCL VE-cadherin and untreated control cells. Mean±s.e.m. from four independent experiments. (B,D) **P≤0.005, ***P≤0.001; ns, not significant.

BMP6 induces phosphorylation of VE-cadherin via activation of c-Src. (A) VE-cadherin is phosphorylated at Tyr685 upon BMP6 treatment. VE-cadherin was immunoprecipitated with a VE-cadherin-specific antibody from confluent HUVECs treated with BMP6 for 30 min and samples were blotted as indicated. Normal IgG antibody served as the immunoprecipitation (IP) control. TCLs represent lysates not subjected to IP. Dotted lines indicate where samples from the same blot have been omitted. (B) Quantitation of pVE-cadherin_Tyr685 signal intensities depicted in A and normalized to TCL VE-cadherin and untreated control cells. Mean±s.d. from four independent experiments. (C) c-Src kinase is activated upon BMP6 treatment. c-Src was immunoprecipitated with a c-Src-specific antibody from confluent HUVECs treated with growth factors for 15 min. Normal IgG antibody served as an IP control. (D) Quantitation of pSrc_Tyr416 signal intensities shown in C and normalized to TCL c-Src and untreated control cells. Mean±s.d. from three independent experiments. (E) BMPRII interacts endogenously with c-Src. BMPRII was immunoprecipitated with a BMPRII-specific antibody from confluent HUVECs stimulated with BMP6 for 30 min and samples were blotted as indicated. Quantitation shows the c-Src versus BMPRII IP signal intensity ratio. Normal IgG antibody served as an IP control. (F) ALK2 associates with c-Src. HEK293T cells were transfected with ALK2-HA and c-Src, and HA-tagged ALK2 was immunoprecipitated with an HA-specific antibody. Normal IgG antibody served as IP control. (G) ALK2 and c-Src are required for BMP-induced phosphorylation of VE-cadherin. HUVECs were transfected with siRNA targeting either nonspecific sequences (si-scr), human ALK2 (si-ALK2) or human c-Src (si-SRC) and treated with BMP6 for 30 min. Cells were lysed and blotted as indicated. (H) BMP6-induced permeability is mediated by ALK2 and c-Src. HUVECs were transfected with si-scr, si-ALK2 or si-SRC, seeded in transwell inserts and stimulated with BMP6 for 24 h. At the indicated time points, TEER was measured. Mean±s.d. normalized to untreated control cells from three independent experiments. ***P≤0.001; ns, not significant. For BMP6-treated si-ALK2 and si-SRC versus untreated si-scr: ^##P≤0.005, ^###P≤0.001.

VE-cadherin regulates endothelial BMP signaling. (A) VE-cadherin is required for efficient BMP signal transduction. HUVECs were transfected with si-scr or si-CDH5 and treated with BMP6 for the indicated times. Cells were lysed and blotted as indicated. (B) Quantitation of pSMAD1/5 signal intensities shown in A and normalized to GAPDH and untreated control cells. Mean±s.d. from three independent experiments. (C) VE-cadherin is required for optimal activation of SMAD1/5 target gene expression. HUVECs were transfected with si-scr or si-CDH5 and treated with BMP6 for 60 min. ID1 mRNA expression was determined using qRT-PCR and normalized to GAPDH control mRNA expression and to untreated control cells. Mean±s.e.m. from three independent experiments. (D) CDH5 mRNA expression was determined after siRNA transfection using qRT-PCR and normalized to GAPDH control mRNA expression and to si-scr-treated control cells. Mean±s.e.m. from three independent experiments. (E) VE-cadherin clusters are needed for efficient BMP signaling. HUVECs were incubated with the VE-cadherin extracellular domain-targeting antibody BV6 or isotype control antibody and treated with BMP6 for 45 min. Cells were lysed and blotted as indicated. (F) Quantitation of pSMAD1/5 signal intensities depicted in E and normalized to GAPDH and untreated control cells. Mean±s.d. from three independent experiments. **P≤0.005, ***P≤0.001. For BMP6-treated si-scr and si-CDH5: ^#P≤0.05, ^###P≤0.001.

VE-cadherin interacts with BMP receptors. (A) In situ proximity ligation assay (PLA) of VE-cadherin and the BMP receptors ALK2 and BMPRII. HUVECs were treated with BMP6 for 60 min and association of VE-cadherin and ALK2 or BMPRII was visualized using an in situ PLA (green). DAPI, blue. Scale bars: 10 µm. (B) Quantitation of VE-cadherin–ALK2 and VE-cadherin–BMPRII heteromers shown in A. Mean±s.d. from three independent experiments. (C) VE-cadherin interacts endogenously with BMPRII. VE-cadherin was immunoprecipitated with a VE-cadherin-specific antibody from confluent HUVECs stimulated with BMP6 for 60 min and samples were blotted as indicated. Recombinant protein A-Sepharose beads served as an IP control. TCLs represent lysates not subjected to immunoprecipitation. (D) VE-cadherin associates with BMPRII upon overexpression. HEK293T cells were transfected with VE-cadherin and BMPRII-HA, and VE-cadherin was immunoprecipitated with a VE-cadherin-specific antibody. Normal IgG antibody served as IP control. (E) VE-cadherin associates with ALK2 upon overexpression. HEK293T cells were transfected with VE-cadherin and ALK2-HA, and VE-cadherin was immunoprecipitated with a VE-cadherin-specific antibody. Normal IgG antibody served as IP control. (F) VE-cadherin facilitates BMP receptor complex formation. HEK293T cells were transfected with BMPRII-Myc and ALK2-HA in the absence or presence of co-expressed VE-cadherin. Cells were stimulated with BMP6 for 45 min, followed by immunoprecipitation of Myc-tagged BMPRII with a Myc-specific antibody. Mouse IgG isotype antibody served as IP control. **P≤0.005, ***P≤0.001.